Methods for the detection of urinary proteins are often more sensitive to albumin than to other urinary proteins. However, it is important to detect proteins other than albumin, especially in the case of Bence-Jones proteinuria. The detection of proteins should cover a wide range of protein concentration since the decision levels and recommended actions to be taken by clinicians will vary depending on the concentration of protein detected in a patient's urine. For example persistent proteinuria greater than 50 mg/dL represents strong evidence of renal disease whereas a protein level of greater than 300 mg/dL is consistent with a diagnosis of nephrotic syndrome. A concentration of protein in urine of greater than 800 mg/dL suggests massive protein loss and warrants a renal biopsy and/or steroid therapy. Accordingly, it is apparent that a test for protein in urine should be effective over a wide range of protein values.
Various methods for the determination of protein in aqueous fluid have been reported in the literature. These methods include the Kjeldahl methods, biuret method, Lowery method, dyestuff combination method, UV method and fluorometric method. In general, proteins react with various substances, particularly with dyes such as bromphenol blue, coomassie brilliant blue and eosine as well as metal ions such as silver (I), copper (II), zinc (II) and lead (II). Typically, the addition of protein to the reaction between a dye and a metal ion results in a spectral change in the dye-metal ion solution. Fujita et al report in Bunseki Kagaku Vol. 32, Pp. E379-E386 that the addition of protein to the reaction between pyrogallol red and molybdenum (VI) produces a different spectrum than that of the pyrogallol red-molybdenum(VI) complex solution. Japanese Kokai Patent No. SHO 62 1987!-6170 discloses a test strip for protein determination comprising a molybdate salt, a pigment which forms a complex with molybdate and whose absorption band is shifted in the presence of a protein together with a chelating agent which combines with molybdate ions. A similar assay for trace amounts of protein is disclosed in Japanese Kokai Patent 61-155757 in which there is described the use of a chelating agent which is able to bond with molybdenum or a metal ion which is able to bond with oxalic acid, citric acid, phosphoric acid or salts thereof normally present in the urine test sample. This assay is a dye binding method using the complex of pyrogallol red and molybdenum. At low pH the dye-metal complex is red. The color changes to blue when deprotenated at higher pH. The protein causes the dye to deprotenate more easily (at a lower pH) by the interaction of positively charged amino acid groups stabilizing the negatively charged deprotenated dye-molybdate complex.
It has more recently been discovered that tungstate behaves in a manner similar to molybdate in the presence of protein and a dye. Useful molybdate and tungstate salts for the total protein assay of the present invention include sodium, potassium, lithium and ammonium salts or a tungstate/molybdate with an alkyl, dialkyl, trialkyl or tetraalkylammonium ion or a phosphotungstate bearing a similar cation.
In U.S. Pat. No. 5,399,498 there is disclosed the use of certain ionizable phosphate containing compounds to reduce background reactivity in the protein assay which uses a molybdate or tungstate salt and a dye which forms a complex with molybdate or tungstate whose absorption band is shifted in the presence of protein. This reference does not, however, demonstrate that the method can operate over the total range of protein concentrations. U.S. Pat. No. 5,374,561 discloses that nitroso substituted polyhalogenated phenol sulfonephthaleins can be used to detect albumin but does not demonstrate that this dye class can be used in combination with a pyrogallol dye. There is disclosed in U.S. Pat. No. 5,279,790 an analysis method whereby nitro substituted polyhalogenated phenolsulfonephthaleins can be combined with a merocyanine dye to detect albumin.
It would be desirable, and it is an object of the present invention to provide a protein assay based on the interaction of molybdate or tungstate and pyrogallol red which operates over a wide range of protein concentrations.